A MUTATOR AFFECTING THE REGION O F THE ISO-1-CYTOCHROME c GENE IN YEAST SUSAN W. LIEBMAN,1 ARJUN SINGH2
AND
FRED SHERMAN3
Department of Radiation Biology and Biophysics, University of Rochester School of Medicine and Dentistry, Rochester, N e w York AND
Department of Biological Sciences, Uniuersity of Illinois at Chicago Circle Box 4348, Chicago, Illinois 60680 Manuscript received August 8,1977 Revised copy received January 23,1979 ABSTRACT
The mutator gene DELI in the yeast Saccharomyces cereuisiae cauces a high rate of formation of multisite mutations that encompass the following three adjacent genes: CYCI, which determines the structure of iso-l-cytochrome c; RAD7, which controls UV sensitivity; and O S M I , which controls osomotic sensitivity. The simplest hypothesis is that these multisite mutations are deletions, although it has not been excluded that they may involve other types of gross chromosomal aberrations. In contrast, normal strains do not produce such multisite mutations even after mutagenic treatments.--The multisite mutations arise at a rate of approximately 1 0 - 6 to 10-6 per cell per division in DELI strains, which is much higher than rates observed for mutation of genes in normal strains. For example, normal strains produce all types of cycl mutants a t a low rate of approximately 10-8 to 10-9. No evidence for multisite mutations was obtained upon analysis of numerous spontaneous adel, ade2, met2 and met15 mutants isolated in a DELI strain. DELI segregates as a single Mendelian gene closely linked to the CYCI locus. DELI appears to be both cis- and tram-dominant. The location of the DELI gene and the lack of effect on other genes suggest that the mutator acts only on a region adjacent to itself.
is now clear that genetic factors within an organism exert considerab'lecon;'rol on spontaneous mutagenesis. Mutators and antimutators that affect the types and frequencies of mutations occurring throughout the genome have been described in viruses, bacteria, yeast and other eukaryotes. In addition to the general mutators, specific genes with a high spontaneous mutation rate, referred to as mutable genes, have been extensively studied in bacteria, maize and Drosophila (see GENETICSSupplement 1973, 73: 1-205 for review). Aside from the mutable genes, the occurrence of extensive genetic deletions or gross chromosomal aberrations is a rare phenomenon. In yeast, such aberraPresent address: Department of Biological Sciences, University of Illinois at Chicag, Circle, Chicago, Box 434.8, Chicago, Illinois 60680. Present address: Department of Biochemistry, University of Wisconsin, Madison, Wisconsin 53706. Present address: Department of Radiaiton Biology and Biophysics, University of Rochester, School of Medicine and Dentistry, Rochester, New York. Genetics 92: 783-802 July, 1979.
784
S. LIEBMAN, A. SINGH A N D F. SHERMAN
tions comprise at most a few percent of all forward mutations. Until recently, the iso-1-cytochrome c (CYCl) locus was no exception. Of 353 spontaneous and induced cycl mutants selected from the same parent strain, D311-3A, none contained extended deletions or gross chromosomal aberrations of the CYCl locus (SHERMAN et al. 1974; SHERMAN, unpublished results). Two other iso-1-cytochrome c mutants, cycl-l and cycl-237, were uncovered during examination of strains in studies that did not involve direct selection procedures. Both of these alleles were multisite mutations. They can not be reverted by treatment with a variety of mutagens, and they do not recombine with a large and SHERMAN number of cycl point mutants that cover the entire gene (PARKER 1969; SHERMAN et al. 1974; SHERMAN, unpublished results). Furthermore, two unusual properties segregate with cycl-1, but not with revertible cycl alleles, namely sensitivity to UV and inhibition of growth by high osmotic media (SINGHand SHERMAN,1978). The radiation and osmotic sensitivities associated with cycl-l arise, respectively, because the mutation encompasses a t least part of the neighboring rad7 and osml loci (SINGHand SHERMAN,1978). The coincidence that the only multisite cycl mutations were the two cycl mutants uncovered unintentionally remained unexplained until the discovery of the DELl gene described in this paper. It now appears that these gross chromosomal aberrations in the cycl regions may have arisen from strains carrying specific mutators similar or identical to D E L l . Numerous other multisite mutations presumed to be deletions that cover various regions of the cycl gene have recently been detected in specifically designed et al. 1975). Diploid strains were constructed with extenexperiments (SHERMAN sive dissimilarities of sequence in homologous regions of the CYCl gene, using mutants having functional, but altered. iso-1-cytochrome c. These strains were sporulated and mutants having deficieiicies of iso-1-cytochrome c were selected from the meiotic progeny. It was speculated that mispairing due to extensive differences in homologous regions of the C Y C l gene might lead to deletions during meiosis. Over one-fourth of the mutants from some crosses contained deletions of various lengths, ranging from those covering only two adjacent sites to those covering at least the entire CYCl gene. Surprisingly, the heteroallelic regions were not necessarily deleted, since approximately 40% of the deletions did not encompass the altered segment of the parent strains. At the present time, it is not clear whether the majority of the deletions in this study did indeed arise as the result of heteroallelic mispairing. Alternatively, genetic factors similar to the DELI gene described in this paper could have been partially responsible for the generation of the deletions obtained in the study of SHERMAN et al. (1975). The present paper deals with a mutator gene, D E L l , which causes a high frequency of multisite mutations in the region 01the CYCl locus. During the course of isolating cycl mutants from a variety of strains, we uncovered an unusual strain, D609-2A, which produced high frequencies of cycl mutants. Over 95% of these cycl mutants contained mutations that encompassed the entire CYCl locus and extended into the adjacent RAD7 and OSMl loci. The simplest hypothesis is that these mutations are deletions, but the possibility that
A MUTABLE GENE I N YEAST
785
they involve other gross chromosomal aberrations has not been excluded. The studies described here establish that the mutable property associated with D609-2A is under the control of a Mendelian gene denoted DELI. The genetic and phenotypic properties of this unusual gene are described. MATERIALS AND METHODS
Genetic nomenclature: Dominant alleles are indicated by capital letters, while recessive alleles are denoted by lower case letters. CYCI+ refers to the wild-type allele controlling the structure of ]so-I-cytochrome c. Mutant alleles of this gene are denoted cycl-I, CYCI-2, etc. An intragenic revertant of the cycl-I66 mutant is designated CYCI-166-B ( SCHWEINGRUB~, SHERMAN and STEWART 1977). SUP4-o refers to one of the eight UAA suppressors that cause the insertion of tyrosine at ochre (UAA) sites (GILMORE, STEWART and SHERMAN 1971). Media: General types of media and media used specifically with the iso-I-cytochrome C system have been described in detail by SHERMAN et al. (1974). Chlorolactate medium (1% Bacto-yeast extract, 2% Bacto-peptone, 3% (V/V) glycerol, 1% Ionagar, and 0.05% j3-chlomlactic acid) is used to enrich for cycl mutants. Lactate medium (0.67% Bacto-yeast nitrogen base without amino acids, 0.05% Difco yeast extract, 1.5% Ionagar and 1% DL-lactate) is used to select for CYCI revertants or CYCI recombinants. Canavanine sulfate (60 mg/l) was added to synthetic medium lacking arginine for scoring can1 mutants. Hypertonic media as described by SINGHand SHERMAN(1978) (1% Bacto-yeast extract, 2% Bacto-peptone, 2% dextrose, 2% Ionagar, 2.5 M ethylene glycol o r dextrose) was used to score for osml mutants. General genetic methods: Standard yeast genetic procedures of crossing, sporulation and tetrad analysis were generally used to construct tester strains and analyze gene segregations. Since the SUP4-o and D E L l markers are closely linked, the construction of the DELI CYCI+ SUP4-o strain required the introduction of the SUP4-o marker by mutation rather than by conventional meiotic recombination. UAA suppressors were conveniently isolated in the haploid strain SL26O-iB (DELI CYCl+ lysl-I his5-2) by selecting for simultaneous revision of the UAA markers lysl-I and his5-2. Most of these revertants contained a UAA suppressor. To distinguish which of these suppressors were SUP4-0, we used the test devised by HAWTHORNE and MORTIMER (1968) to determine if two dominant suppressors are a t the same locus. The revertants were crossed to a tester containing SUP4-o and a UAA-suppressible allele conferring canavanine resistance (canl-100). The diploids were sporulated and spotted on a medium containing canavanine. Only segregants lacking suppressors permit expression of the canavanine-resistant allele. Thus, the absence of growth on the canavanine media indicated that the suppressor i n question was allelic to SUP4. The genotype of the SUP4-o D E L f stain used in this study was verified by tetrad analysis, which demonstrated that the suppressor was closely linked to CYCI. Scoring methods: Nutritional markers were scored by spotting cell suspensions on appropriate media. The SUP4-o ochre suppressor (GILMORE1967; GILMORE,STEWART and SHERMAN1971) was scored by suppression of UAA alleles. The rad7 gene (LAWRENCE et al. 1975), causing UV sensitivity, was scored by spotting cell suspensions on complete media and irradiating with 120 Jm-2. The osml gene (SINGH and SHERMAN1978), causing osmotic sensitivity, was scored by spotting cell suspensions on hypertonic media. The CYCl+ normal strains, the cycl strains lacking iso-I-cytochrome c and the CYCI-166-B strains containing an abnormal c,-band (SCHWEINGRUBER, SHERMANand STEWART1977) were distinguished by low-temperature (-190") spectroscopic examination of intact cells (SHERMAN and SLONIMSKI 1964). Isolation of cycl mutants: Cycl mutants were isolated by the chlorolactate selection technique previously described by SHERMAN et al. (1974). In comparison to CYCI + normal strains, the cycl mutants grow poorly on lactate medium, but better on chlorolactate medium. From IO6 to 10' washed cells were spread on each chlorolactate plate. The plates were incubated from five to ten days and resistant colonies tested. Depending upon the parent strain, from zero to 100% of these chlorolactate-resistant mutants were unable to grow on lactate media. The cycl
786
S. LIEBMAN, A. S I N G H A N D F. SHERMAN
mutants among these were identified by their inability to complement cycl tester strains. In addition, the cytochrome c contents of presumptive cycl mutants were determined by low temperature spectroscopy. Isolation of met2 and met15 mutants: Spontaneous met2 and met15 mutants were isolated on the basis of their resistance to methyl mercury (SINGHand SHERMAN 1974, 1975). The two loci were distinguished by complementation tests. Isolation of adel and ade2 mutants: The adel and ade2 mutants were isolated by using their red o r pink morphology to distinguish them from white normal colonies. The frequency of these mutants in cultures to be screened was increased by the nystatin enrichment technique (SNOW 1966). The treatment conditions in the first 12 experiments were varied to search for optimal conditions. The specific conditions for the later 38 experiments were as follows. Fresh stationary phase cultures of SL35&4B (a his1 D E L I ) were washed and suspended in nitrogen starvation msdium (0.67% yeast nitrogen base without amino acids o r ammonium sulfate, 2% dextrose, and 20 mg/l histidine) at a density of 1.5 x 108 to 1.8 x 108 cells per ml. The cultures were starved overnight 15 to 20 hr) and then incubated for 3.5 h r i n minimal medium supplemented with histidine. Nystatin was added to a final concentration of 20 fig per ml. At this stage, prototrophic cells had begun growth, while auxotrophs were still not dividing. Nystatin selectively kills growing cells and thus enriches for auxotrophs. After one hr, cells were collected by centrifugation, washed twice with distilled water and plated on minimal medium supplemented with histidine and adenine (10 mg/l). A reduced amount of the normal concentration of adenine was used for enhanced color development by adenine auxotrophs. Nystatin was first dissolved in N, N-dimethyl-formamide (2 mg/ml), which was then diluted ten-fold in sterile water. Then 0.3 ml of this solution was added to 2.7 ml of cell suspension. An average of 7.5 x 10.7 treated cells were screened for adel and ade2 mutants i n each of the 38 experiments. The survival in 36 experiments varied from 10-5 to 2 x 10-6, while i n the other two experiments it was IW. The red and pink colonies were isolated, tested for adenine requirement and classified as adel or ade2 by complementation tests. I?ilutagenic treatments: (a) EMS. Cells were treated with 3% ethyl methanesulfonate (EMS) i n 0.1 M sodium phosphate buffer (pH 7.2) for one h r at 30" and were then directly plated on selective media. (b) X rays. Cells were irradiated on the surface of plates with 25 krad by means of an OEG-60-T Machlett X-ray tube powered by a custom made X-ray generator (Picker Corp ). (c) UV Cells on the surfaces of the plates were irradiated with 62.4 Jm-2 of UV. Characterimtion of cycl mutat'ons: The nature of the unknown cycl mutants was tested by crossos to two or three well-characterized point mutants, described in Table 1, that span the TABLE 1 T h e cycl point-mutnnt tester alleles Allele
cy c l -I 3 cycI-31 cycl-6
Amino acid position'
____
-1
3 12
cycl-2
21
cycz-72
66
cycl -76 cycl-I23
71 (l02?)
Reference
STEWART et al. 1971 SHERMAN and STEWART 1973,1974 PUTTERMAN, MARGOLIASH and SHERMAN 1974 STEWART et al. unpublished; (see SHERMAN and STEWART 1974) STEWART et al. unpublished; (see SHERMAN and STEWART 1974) STEWART and SHERMAN 1973 SHERMAN et al. 1975
* The numbers refer to the amino acid positions of the corresponding mutational sites that were determined from altered iso-1-cytochromes c in revertants. The cycl-I23 site has been estimated from fine-structure mapping.
787
A MUTABLE GENE I N YEAST
entire gene. The X-ray-induced mitotic recombination frequencies of the heteroallelic diploids were then determined. The transfer of cell suspensions for crossing and testing was facilitated et al. (1974). Cell suspensions of the by the use of inoculating rods as described by SHERMAN heteroallelic diploids were spotted on several lactate plates, and the surfaces of one or two sets of plates were irradiated with 5 krad. The lactate plates were scored after five days of incubation a t 30". Recombination was indicated when increased numbers of colonies appeared over the spotted inocula that were irradiated in comparison to the unirradiated controls. Lack of colonies indicated that recombination did not occur or occurred a t a low rate. A more quantitative measure of recombination rates was occasionally undertaken to verify the spot-test results. Cell suspensions were conveniently prepared free of growth medium and a t a known titre by inoculating the surfaces of 0.5 ml of medium (0.4% Bacto-yeast extract, 0.8% Bacto-peptone, 0.8% glucose and 1% Ionagar) in one-dram vials and, after three days of incubation, suspending the cells in 2 m l of sterile water (SHERMANet a2 1974). Lactate plates were each spread with 0.2 m l of the suspensions from these vials, which contained approximately 1.2 X 107 cells, and two sets d plates were irradiated with 5 krad. The irradiated and unirradiated plates were scored after incubation as indicated above. Characteiization of mete, metl5, ade2, and adel mutants: A similar procedure was used to test for intragenic recombination between unknown met2, m e t l 5 , a h 2 and a d d mutants and appropriate point-mutant tester strains. Although these testers are not as well characterized as the c y c l tester strains, we attempted to choose mutants that would span much of the length of the genes. For m e d 5 , this was done on the basis of previous fine-structure or intragenic complementation maps (SINGHand SHERMAN 1975). RESULTS
The origin and classification of the DELI mutator: During the course of experiments involving the selection of cycl mutants from a variety of strains by the chlorolactate procedure, it was noted that strain D609-2A produced an unusually high frequency of spontaneous cycl mutants. In addition, further investigation revealed that over 95 % of the cycl mutants arising from this and related strains contained multisite mutations encompassing the entire cycl locus; in contrast, such mutations occurred in less than 5 % of the cycl mutants arising from normal strains. Genetic analysis described below demonstrated that the high rate of production of these multisite mutations can be attributed to a single dominant gene designated D E L l . Table 2 summarizes the analysis of a total of 870 spontaneous cycl mutants kolated from 65 different normal (dell+) and mutator ( D E L I ) strains. The cycl mutants were obtained with chlorolactate selection (see MATERIALS AND METHODS) and were classified as either containing or not containing multisite mutations by the various techniques described below. TABLE 2
Isohtion and characterization of cycl mutants f r o m normal and DELI haploid strains Normal dell+
No. haploid strains examined No. c y c l mutants tested No. cycl multisite mutations Percent multisite mutations
28 355 9 3%
Mutator
DELI
37 515 5(17 98%
788
S. L I E B M A N , A. S I N G H A N D F. S H E R M A N
In the early studies, all cycl mutants were actually tested for intragenic recombination with two or three cycl point mutants that map at known positions spanning the entire cycl gene (see MATERIALS AND METHODS). Those mutants that failed to recombine with all of the testers were classified as multisite mutations covering the entire cycl locus. Those mutants that showed recombination were considered to be point mutants or small deletions. Using this classification method, only four multisite mutations were obtained among the 234 cycZ mutants isolated from 14 normal dell+ strains. In contrast, a total of 182 multisite mutants covering the entire cycl locus were found among the 185 cycl mutants isolated from 17 DELI strains. Each of these cycl mutants were also examined for UV sensitivity to see if the mutations extended into the neighboring rad7 locus (see MATERIALS AND METHODS). As expected, none of the “point” mutants were UV sensitive. In contrast most (183 out of 186) of the multisite mutations were sensitive to U V . Furthermore, all of the 182 multisite mutations that arose in DELI strains were UV sensitive, while only one of the four multisite mutations isolated in normal dell + strains was sensitive to UV. In order to further define the size of these multisite mutations, complementation tests with the nearby osml gene were undertaken with a representative number of UV-sensitive multisite mutants (see MATERIALS AND METHODS). All 51 of the UV-sensitive multisite mutants examined, including the one isolated from a normal strain, failed to complement the osml tester. This indicates that all of these mutations extend into at least part of the O S M l locus. In contrast, the three multisite mutants that were not UV sensitive and that were isolated from normal strains complemented osml and therefore did not extend into the OSMl gene. It appears that cycl mutations that arise in DELI strains all extend into at least part of the neighboring RAD7 and OSMl loci. I n contrast, the occasional multisite mutations obtained from normal strains seem to vary in length and do not necessarily include any part of RAD7 or OSMl. These results suggested a simpler method to distinguish the multisite mutations produced in DEL2 strains from the “point” cycl mutants that arise in normal strains. Thus in the subsequent examination of 451 spontaneous cycl mutants isolated from 34 normal and DELI haploid strains, all cycl mutants were tested for UV and osmotic sensitivity. Those cycl mutants that concurrently gained the unselected characteristics of sensitivity to UV and hypertonic medium were considered to contain multisite mutations. The cyci mutants that did not gain these sensitivities were classified as “point” mutants. The classifications of representative cycZ mutants isolated from each parent strain were then further checked by interallelic recombination. I n this way, 325 of 330 cycl mutants isolated from 20 DEL2 strains were found to contain extensive multisite mutations. Of the I21 cycl mutants isolated from 14 normal strains, none contained multisite mutations covering the C Y C I , RAD7 and OSMZ loci. However, five of these mutants were multisite mutations that covered the entire cycl gene, but that did not extend into the RAD7 or OSMZ loci.
A MUTABLE GENE IN YEAST
789
In all of these studies, the distinction between DELI and normal dell+ strains remained clear. In DELI strains, multisite mutations constitute the majority of all spontaneous cycl mutants. These mutations completely encompassed the cycl gene and extended into the neighboring rad7 and osml genes. In dell+ strains, multisitp mutations constitute less than 5% of all spontaneous cycl mutants. The occasional multisite mutations that do arise in dell+ strains do not necessarily cover the RAD7 and OSMl loci. Detailed characteristics of multisite mutations arising in representative DELl strains: To ensure that thc numerous rnultisiie mutations arising in DEL2 strains resulted hom independent events, the chlorolactate procedure was used to isolate cycl mutants from 50 independent subclones of the DELI strain, SL356-4B. One cycl mutant from each subclone was crossed with the three tester strains cycl-23, cycl-2 and cycl-123, which define alterations at positions -1, 21 and 102, respectively, in the 108-residue iso-I-cytochrome c protein. No intragenic recombination was observed in any of these crosses by either the spot or plate tests described in MATERIALS AND METHODS. In addition, each of the 50 mutants failed to complement rad7 and osml strains. Thus, all 50 cycl mutants isolated independently from SL356-4B were multisite mutations that covered the entire cycl gene and exlended into at least part of both the rad7 and osml loci. It was more difficult to determine whether the multisite mutations arising from DEL2 strains also extended into SUP4, anoither gene that is closely linked to cycl (LAWRENCE et al. 1975). The difficulty arises because SUP4 is dominanh so that its inclusion in the mutations could not be established by a simple complementation test. Thus, the approach used was to isolate cycl mutations in DELI stains that contained the UAA suppressor SUP4-o (see MATERIALS AND METHODS for the procedure used to construct the strain). It was reasoned that cycl multisite mutants covering the SUP4 locus would cause the loss of activity of the UAA suppressor. In several multisite mutants, the suppressor activity was lost; in other multisite mutants, the suppressor activity was retaind. These results prove that the multisite mutations do not always cover SUP4. However, since the SUP4-o marker was found to be unstable on chlorolactate medium, it is not clear whether any of the multisite mutations really cover the SUP4 locus. Anather approach to determining whether SUP4 is encompassed by the multisite mutations arising in DELI strains was to' select for loss of the UAA-suppressor activity in a DELI SUP4-o strain and to see if any other neighboring genes were also lost, thus indicating the presence of multisite mutations. This test was accomplished with the help of a UAA marker, cad-100, also present in the DEL? SUP4-o strain. The cad-100 marker ordinarily causes resistance to canavanine; holwever, in the presence of SUP4-0, the canl-100 marker is suppressed and the strain is sensitive to canavanine. Thus, by selecting for revertants that are resistant to canavanine, we were able to select for lass of the SUP4-o Suppressor. Out of 30 revertants that had lost the SUP4-o p h e n w e , none were UV or osmotic sensitive. Thus, there is no evidence that the SUP4 revertants induced in the DELI strain contained multisite mutations. To further characterize the nature of the multisite mutations, we attempted
790
S. LIEBMAN, A. SINGH A N D F. SHERMAN
to revert a representative multisite mutation. In order to eliminate the occurrence of recessive mutations that cause growth on lactate medium by increasing the et al. 1974), tests of revertibility were level of iso-2-cytochrome c (SHERMAN performed on a diploid strain homozygous for one of the multisite mutations generated in a DELI strain. The diploid strain was treated with UV, X rays and and approximately 4.4 X 10' EMS as described in MATERIALS AND METHODS, treated and untreated cells were plated on lactate medium. No revertants were observed, which is consistent with the conclusion that the multisite cycl mutants arising from DELl strains are extensive deletions or are other gross chromosomal aberrations. Action of DELl on other genes: Experiments were undertaken in order to determine whether DELI is a general mutator that can act on various regions of the yeast genome. Taking advantage of the red color of adel and ade2 mutants and of the methyl mercury resistance of met2 and met15 mutants, and using the nystatin enrichment technique, spontaneous mutants were isolated in the DELI strain, SL3564B, and examined for the nonrevestible, multisite mutations. As described above, 50 independently derived cycl mutants isolated frcm different subclones of SL3564B were all found to contain multisite mutations of the cycl region. In contrast, none of the five met2, 31 metI5, 26 a b 2 and 31 adel independent mutations isolated from this DELI strain were classified as multisite mutants. Each of these mutants, except for two that were too leaky to1 be tested, showed either intragenic recombination with tester strains or could be reverted to the apparent wild-type state. Thus, there is no evidence that DELl acts on regions other than the region encompassing the CYCI locus. However, the possibility that DELI can act on regions other than those tested or that multisite mutations produced at the ade acd met loci were not detected because they are lethal can not be eliminated. Rates of adel arid ade2 mutations: As part of the comparative study of spontaneous mutation rates in DELI strains, the rates of adel and ade2 mutations were calculated from data of the last 38 nystatin enrichment experiments per€ormed on the DELI strain, SL3564B (see MATERIALS AND METHODS for experimental details). Mutation rates, a, were obtained from the equations Po," ln(m) = 1.24 and a = m (ln2)/N (LEAand COULSON 1949) and from r = m ln(Cm) and a = m (ln2)/N (LURIAand DELBRUCK 1943), where: a = mutation rate (mutations per cell per generation) ;N = final number of cells per culture; m = the number of mutations per culture; C = number of cultures; I, = the number of mutants in the median culture; and r = t h c average number of mutants per culture among C cultures. In the calculations, we used our experimental values of C=38, N=7.5 x l o 7 and the observed median ( r , ) and average ( r ) number of adel and ade2 mutants per culture. The experimental values of r and r, and the calculated values of a are shown in Table 3. The results indicate that the spontaneous forward mutation rates to adel and ade2 in the DELI strain, SL356-4B, are approximately 5 x 10-9 to 8 X 1P9. Rates of cycl mutation in DELl and normal strains: The forward mutation rates to cycl in the DELI strains SL356JI.B and SL392-2B were compared with
791
A MUTABLE GENE IN YEAST
TABLE 3 Forward mutaticil rates to adel and ade2 in the DELI strain, SL356-4B r
adel ade2
1.82 1.55
a*
5.5 x 10-9 x 10-9
4.8
nt
To
1 1
8.3 8.3
x 10-9 x 10-9
* a was calculated from the formulas r = m ln(Cm) (LURIAand DELBRUCK 1943) and a = m (ln2)/n, where N = 7.5 x IO7 and C = 38. + a was calculated from the formulas r , / m - ln(m) = 1.24 (LEAand COULSON1949) and a = m (ln2)/N, where N = 7.5 x 107.
the cycl mutation rates in the normal strains D311-3A and D911-2B. A variety of methods were used to estimate these rates. The mutation rate to cycl in D911-2B was obtained using the Luria-Delbruck (LURIAand DELBRUCK 1943) fluctuation test. D911-2B was subcloned on complete media, and 55 colonies, each containing l o s cells, were individually suspended in water and spread o n 9-cm diameter agar plates containing chlorolactate medium. Henceforth, the number of cells per plate was determined by quantitatively washing cells off the plates. Within two days, the number of cells per plate increased to 1.8 x 1Os, where it remained constant for an additional several days until the resistant colonies began to appear. Chlorolactate-resistant colonies were then picked and tested to determine if they were cycl mutants. As soon as one cycl mutation was found from a given plate, additional chlorolactate-resistant mutants from that plate were no longer tested. Plates that did not give any cycl mutations were picked and tested exhaustively. Of the 65 cycl mutations obtained from D911-2B, less than 5% were multisite mutations. Among the 55 plates examined, 16 did not contain any cycl mutants, while 39 plates contained one or more cycl mutants. Using the equations po = cmand a = m (ln2)/Nt-N0 LURIAand DELBRUCK (1943), where p. is the fraction of plates that do not have any cycl mutants and is equal t o 0.29 in the present experiment; m is the average number of cycl mutations that occurred per culture; N t is the final cell count per plate and is equal to 1.8 f 0.1 X lo8 in the present experiment; N o is the initial number of cells per colony on complete media and is equal to one in the present experiment; and a is the mutation rate, mutations per cell per generation, we calculate the mutation rate to be 4.8 x l k9. Attempts to use the same fluctuation methold to measure the mutation rate in DELI strains failed because all of the plates contained cycl mutants, apparently due to an increased mutation rate. We were unable to reduce the number of colonies because the background growth led to a minimum of 1.1 x lo8 cells per 9-cm diameter plate, no matter how many cells were initially placed on the plate. Even when 1.4-cm diameter wells were used instead of plates, background growth led to a titer 04 approximately 2 x lo6 cells per well, and all of 54 wells contained one or more cycl mutant colonies. Because of these problems, the mutation rate in the DELI strain SL392-2B was measured by an adaptation of the LURIAand DELBRUCK (1943) method. To determine the mutation rate, l o 4
792
S. LIEBMAN, A. SINGH A N D F. SHERMAN
cells from an overnight culture grown in synthetic lactate medium were spread on each of 37 agar wells containing chlorolactate medium. Due to the low frequency of cycl mutants in the culture, it is likely that none of the cells plated were cycl mutants. Within three days, the number of cells per well increased to 1.6 0.4 x IO6, where it remained constant for an additional several days until resistant colonies began to appear. Thus, each cell gave rise to a microcolony containing on the average 160 cells. After further incubation, an average of 6.8 rf: 2.5 cycl mutant colonies arose on each well. Over 95% of these mutants were of the multisite type. Since virtually all of the mutants arose during the formation of the microcolonies, po = 1-6.8 x IO-* and m is approximately equal to 6.8 x Therefore a, the mutation rate per cell per division, was deduced to be approximately 2.9 1.7 x In another approach, mutation rates were estimated by using the principle that when a population equilibrium is reached between mutant and normal cells during exponential gromh in a medium totally selective against the mutant, the mutant frequency is then approximately equal to the mutation rate (OGURet al. 1959). These conditions were met by growing strains in liquid lactate medium where normal CYCl+, but not cycl mutant, cells can grm. Thus, the mutation rates of cycl were simply determined from the frequencies of cycl mutants in an equilibrium population of cells grown in lactate medium. The frequencies o€ cycl mutants were estimated using the chlorolactate selection procedure. The results of several experiments with normal and DEL2 strains are presented in Table 4. The normal dell+ strain, D311-3A, was kept in an exponential phase of growth in lactate medium by periodically transferring cells to fresh medium. After four days of exponential growth, 4.4 x IO6 cells per plate were spread on chlorolactate medium. Within three days, the number of cells per plate increased to 1.1 e 0.1 x lo8. Then the number of cells remained constant for an additional several days until the resistant colonies began to appear. Among the 19 plates examined, a total of 546 resistant colonies appeared. Complementation tests indicated that only 2% of these colonies were cycl mutants. This frequency A indicates that the cycl mutation rate in D311-3A is approximately 5 X
*
*
TABLE 4 Rates of cycl mutations in normal (dell f)and DELl strains Strain X o .
Genotype
Days of growth*
D311-3A D311-3A SL356-4B SL3564.B SL356-4B SL3564B
dell+ dell + DELl DELl DELl DELl
4 7 2 5 5 5
No. of cells per plate:
No. of
3 days
plates
Initial
4.3~106 1.1~108 4.4~106 1 . 1 ~ 1 0 8 1x105 5 . 5 ~ 1 0 5 1.9X107 4.2X107 5.7X106 5.7X1O7 4.7X105 4.7x1O6
19 20 5 3 2
2
Mutants per plate
29 29 6 358 225 40
% cycf mutants
Rate of cycf mutations
2 2 100 100
5x1e9 5x10-Q 11X10-6 9XIW 4X1W 9x10-6
100 100
* Day of exponential growth in liquid lactate medium before plating on chlorolactate plates. The number of cells initially plated increased to approximately 108 per plate due to residual growth. When plating SL3564B, this number was constrained by the addition of other cells.
793
A M U T A B L E G E N E IN YEAST
duplicate determination was made in a similar manner after the exponential culture of D311-3A was grown for an additional three days on lactate medium. None of these mutants were Again the mutation rate was estimated at 5 x multisite mutations. The cycl mutation rate in the DELl strain, SL3564B was estimated by a similar procedure. Here, however, when the background growth of SL3564B on the chlorolactate plates was permitted to reach the limiting level of 1.1 X 10' cells per plate, the resistant colonies were too numerous to score. In order to reduce the background level of SL3564B, various amounts of the normal diploid strain D-261 were added to the chlorolactate plates. These added cells reduce the background growth of SL3564B, since a chlorolactate plate can support growth only to a total of approximately 1-2 x 108 cells. The level of background growth of plates spread with various amounts of D-261 was determined by qwmtitatively washing cells off of the chlorolactate plates and counting them under the microscope. The diploid cells gave rise to virtually no resistant colonies, presumably because most chlorolactate resistant mutations are recessive. In order to measure the cycl mutation frequency in SL356-4B, plates were spread with both D-261 and SL3564B at a ratio of at least, respectively, ten to one. Under these conditions, it is assumed that D-261 will grow to approximately the same level that it reached in the absence of SL356-4.B. It is also assumed that SL356-4.B will increase by the same factor as D-261. The results of four determinations of the cycl mutation rate in the DELl strain SL356-4B are shown in Table 4.The cycl mutation rate is between IO4 and and nearly all of these mutants are multisite mutations. These results, summarized in Table 5, show that the forward mutation rate to cycl is increased by approximately three orders of magnitude in the DELl strains SL392-2B and SL3564B as compared with the normal strains D311-3A and D911-2B. It should be noted that the mutation rate determinations were quite consistent, even though a variety of different strains and methods were used. Since nearly all of the cycl mutants produced in the DELl strains are multisite mutations, while none or very few cycl multisite mutations are produced in normal strains, it is clear that the DELl gene increases the frequency of mutation to cycl multisite mutations by four or five orders of magnitude. In contrast, the forward mutation rates to adel and ade2 estimated in a DELI strain are of TABLE 5 Summary of mutation rate determinations to cycl in normal (dell+) and DELl strains Strain No.
D311-3A D911-2B SL356-4B SL392-2B
Genotype
dell + dell + DELl DELl
Rate of mutations to cycl
5 x 10-9" 4.8 x 10-9t 8 (+. 3) x le6* 2.9 (f1.7) X le6+
Rate of cycl multisite mutations
AND
FRED SHERMAN3
Department of Radiation Biology and Biophysics, University of Rochester School of Medicine and Dentistry, Rochester, N e w York AND
Department of Biological Sciences, Uniuersity of Illinois at Chicago Circle Box 4348, Chicago, Illinois 60680 Manuscript received August 8,1977 Revised copy received January 23,1979 ABSTRACT
The mutator gene DELI in the yeast Saccharomyces cereuisiae cauces a high rate of formation of multisite mutations that encompass the following three adjacent genes: CYCI, which determines the structure of iso-l-cytochrome c; RAD7, which controls UV sensitivity; and O S M I , which controls osomotic sensitivity. The simplest hypothesis is that these multisite mutations are deletions, although it has not been excluded that they may involve other types of gross chromosomal aberrations. In contrast, normal strains do not produce such multisite mutations even after mutagenic treatments.--The multisite mutations arise at a rate of approximately 1 0 - 6 to 10-6 per cell per division in DELI strains, which is much higher than rates observed for mutation of genes in normal strains. For example, normal strains produce all types of cycl mutants a t a low rate of approximately 10-8 to 10-9. No evidence for multisite mutations was obtained upon analysis of numerous spontaneous adel, ade2, met2 and met15 mutants isolated in a DELI strain. DELI segregates as a single Mendelian gene closely linked to the CYCI locus. DELI appears to be both cis- and tram-dominant. The location of the DELI gene and the lack of effect on other genes suggest that the mutator acts only on a region adjacent to itself.
is now clear that genetic factors within an organism exert considerab'lecon;'rol on spontaneous mutagenesis. Mutators and antimutators that affect the types and frequencies of mutations occurring throughout the genome have been described in viruses, bacteria, yeast and other eukaryotes. In addition to the general mutators, specific genes with a high spontaneous mutation rate, referred to as mutable genes, have been extensively studied in bacteria, maize and Drosophila (see GENETICSSupplement 1973, 73: 1-205 for review). Aside from the mutable genes, the occurrence of extensive genetic deletions or gross chromosomal aberrations is a rare phenomenon. In yeast, such aberraPresent address: Department of Biological Sciences, University of Illinois at Chicag, Circle, Chicago, Box 434.8, Chicago, Illinois 60680. Present address: Department of Biochemistry, University of Wisconsin, Madison, Wisconsin 53706. Present address: Department of Radiaiton Biology and Biophysics, University of Rochester, School of Medicine and Dentistry, Rochester, New York. Genetics 92: 783-802 July, 1979.
784
S. LIEBMAN, A. SINGH A N D F. SHERMAN
tions comprise at most a few percent of all forward mutations. Until recently, the iso-1-cytochrome c (CYCl) locus was no exception. Of 353 spontaneous and induced cycl mutants selected from the same parent strain, D311-3A, none contained extended deletions or gross chromosomal aberrations of the CYCl locus (SHERMAN et al. 1974; SHERMAN, unpublished results). Two other iso-1-cytochrome c mutants, cycl-l and cycl-237, were uncovered during examination of strains in studies that did not involve direct selection procedures. Both of these alleles were multisite mutations. They can not be reverted by treatment with a variety of mutagens, and they do not recombine with a large and SHERMAN number of cycl point mutants that cover the entire gene (PARKER 1969; SHERMAN et al. 1974; SHERMAN, unpublished results). Furthermore, two unusual properties segregate with cycl-1, but not with revertible cycl alleles, namely sensitivity to UV and inhibition of growth by high osmotic media (SINGHand SHERMAN,1978). The radiation and osmotic sensitivities associated with cycl-l arise, respectively, because the mutation encompasses a t least part of the neighboring rad7 and osml loci (SINGHand SHERMAN,1978). The coincidence that the only multisite cycl mutations were the two cycl mutants uncovered unintentionally remained unexplained until the discovery of the DELl gene described in this paper. It now appears that these gross chromosomal aberrations in the cycl regions may have arisen from strains carrying specific mutators similar or identical to D E L l . Numerous other multisite mutations presumed to be deletions that cover various regions of the cycl gene have recently been detected in specifically designed et al. 1975). Diploid strains were constructed with extenexperiments (SHERMAN sive dissimilarities of sequence in homologous regions of the CYCl gene, using mutants having functional, but altered. iso-1-cytochrome c. These strains were sporulated and mutants having deficieiicies of iso-1-cytochrome c were selected from the meiotic progeny. It was speculated that mispairing due to extensive differences in homologous regions of the C Y C l gene might lead to deletions during meiosis. Over one-fourth of the mutants from some crosses contained deletions of various lengths, ranging from those covering only two adjacent sites to those covering at least the entire CYCl gene. Surprisingly, the heteroallelic regions were not necessarily deleted, since approximately 40% of the deletions did not encompass the altered segment of the parent strains. At the present time, it is not clear whether the majority of the deletions in this study did indeed arise as the result of heteroallelic mispairing. Alternatively, genetic factors similar to the DELI gene described in this paper could have been partially responsible for the generation of the deletions obtained in the study of SHERMAN et al. (1975). The present paper deals with a mutator gene, D E L l , which causes a high frequency of multisite mutations in the region 01the CYCl locus. During the course of isolating cycl mutants from a variety of strains, we uncovered an unusual strain, D609-2A, which produced high frequencies of cycl mutants. Over 95% of these cycl mutants contained mutations that encompassed the entire CYCl locus and extended into the adjacent RAD7 and OSMl loci. The simplest hypothesis is that these mutations are deletions, but the possibility that
A MUTABLE GENE I N YEAST
785
they involve other gross chromosomal aberrations has not been excluded. The studies described here establish that the mutable property associated with D609-2A is under the control of a Mendelian gene denoted DELI. The genetic and phenotypic properties of this unusual gene are described. MATERIALS AND METHODS
Genetic nomenclature: Dominant alleles are indicated by capital letters, while recessive alleles are denoted by lower case letters. CYCI+ refers to the wild-type allele controlling the structure of ]so-I-cytochrome c. Mutant alleles of this gene are denoted cycl-I, CYCI-2, etc. An intragenic revertant of the cycl-I66 mutant is designated CYCI-166-B ( SCHWEINGRUB~, SHERMAN and STEWART 1977). SUP4-o refers to one of the eight UAA suppressors that cause the insertion of tyrosine at ochre (UAA) sites (GILMORE, STEWART and SHERMAN 1971). Media: General types of media and media used specifically with the iso-I-cytochrome C system have been described in detail by SHERMAN et al. (1974). Chlorolactate medium (1% Bacto-yeast extract, 2% Bacto-peptone, 3% (V/V) glycerol, 1% Ionagar, and 0.05% j3-chlomlactic acid) is used to enrich for cycl mutants. Lactate medium (0.67% Bacto-yeast nitrogen base without amino acids, 0.05% Difco yeast extract, 1.5% Ionagar and 1% DL-lactate) is used to select for CYCI revertants or CYCI recombinants. Canavanine sulfate (60 mg/l) was added to synthetic medium lacking arginine for scoring can1 mutants. Hypertonic media as described by SINGHand SHERMAN(1978) (1% Bacto-yeast extract, 2% Bacto-peptone, 2% dextrose, 2% Ionagar, 2.5 M ethylene glycol o r dextrose) was used to score for osml mutants. General genetic methods: Standard yeast genetic procedures of crossing, sporulation and tetrad analysis were generally used to construct tester strains and analyze gene segregations. Since the SUP4-o and D E L l markers are closely linked, the construction of the DELI CYCI+ SUP4-o strain required the introduction of the SUP4-o marker by mutation rather than by conventional meiotic recombination. UAA suppressors were conveniently isolated in the haploid strain SL26O-iB (DELI CYCl+ lysl-I his5-2) by selecting for simultaneous revision of the UAA markers lysl-I and his5-2. Most of these revertants contained a UAA suppressor. To distinguish which of these suppressors were SUP4-0, we used the test devised by HAWTHORNE and MORTIMER (1968) to determine if two dominant suppressors are a t the same locus. The revertants were crossed to a tester containing SUP4-o and a UAA-suppressible allele conferring canavanine resistance (canl-100). The diploids were sporulated and spotted on a medium containing canavanine. Only segregants lacking suppressors permit expression of the canavanine-resistant allele. Thus, the absence of growth on the canavanine media indicated that the suppressor i n question was allelic to SUP4. The genotype of the SUP4-o D E L f stain used in this study was verified by tetrad analysis, which demonstrated that the suppressor was closely linked to CYCI. Scoring methods: Nutritional markers were scored by spotting cell suspensions on appropriate media. The SUP4-o ochre suppressor (GILMORE1967; GILMORE,STEWART and SHERMAN1971) was scored by suppression of UAA alleles. The rad7 gene (LAWRENCE et al. 1975), causing UV sensitivity, was scored by spotting cell suspensions on complete media and irradiating with 120 Jm-2. The osml gene (SINGH and SHERMAN1978), causing osmotic sensitivity, was scored by spotting cell suspensions on hypertonic media. The CYCl+ normal strains, the cycl strains lacking iso-I-cytochrome c and the CYCI-166-B strains containing an abnormal c,-band (SCHWEINGRUBER, SHERMANand STEWART1977) were distinguished by low-temperature (-190") spectroscopic examination of intact cells (SHERMAN and SLONIMSKI 1964). Isolation of cycl mutants: Cycl mutants were isolated by the chlorolactate selection technique previously described by SHERMAN et al. (1974). In comparison to CYCI + normal strains, the cycl mutants grow poorly on lactate medium, but better on chlorolactate medium. From IO6 to 10' washed cells were spread on each chlorolactate plate. The plates were incubated from five to ten days and resistant colonies tested. Depending upon the parent strain, from zero to 100% of these chlorolactate-resistant mutants were unable to grow on lactate media. The cycl
786
S. LIEBMAN, A. S I N G H A N D F. SHERMAN
mutants among these were identified by their inability to complement cycl tester strains. In addition, the cytochrome c contents of presumptive cycl mutants were determined by low temperature spectroscopy. Isolation of met2 and met15 mutants: Spontaneous met2 and met15 mutants were isolated on the basis of their resistance to methyl mercury (SINGHand SHERMAN 1974, 1975). The two loci were distinguished by complementation tests. Isolation of adel and ade2 mutants: The adel and ade2 mutants were isolated by using their red o r pink morphology to distinguish them from white normal colonies. The frequency of these mutants in cultures to be screened was increased by the nystatin enrichment technique (SNOW 1966). The treatment conditions in the first 12 experiments were varied to search for optimal conditions. The specific conditions for the later 38 experiments were as follows. Fresh stationary phase cultures of SL35&4B (a his1 D E L I ) were washed and suspended in nitrogen starvation msdium (0.67% yeast nitrogen base without amino acids o r ammonium sulfate, 2% dextrose, and 20 mg/l histidine) at a density of 1.5 x 108 to 1.8 x 108 cells per ml. The cultures were starved overnight 15 to 20 hr) and then incubated for 3.5 h r i n minimal medium supplemented with histidine. Nystatin was added to a final concentration of 20 fig per ml. At this stage, prototrophic cells had begun growth, while auxotrophs were still not dividing. Nystatin selectively kills growing cells and thus enriches for auxotrophs. After one hr, cells were collected by centrifugation, washed twice with distilled water and plated on minimal medium supplemented with histidine and adenine (10 mg/l). A reduced amount of the normal concentration of adenine was used for enhanced color development by adenine auxotrophs. Nystatin was first dissolved in N, N-dimethyl-formamide (2 mg/ml), which was then diluted ten-fold in sterile water. Then 0.3 ml of this solution was added to 2.7 ml of cell suspension. An average of 7.5 x 10.7 treated cells were screened for adel and ade2 mutants i n each of the 38 experiments. The survival in 36 experiments varied from 10-5 to 2 x 10-6, while i n the other two experiments it was IW. The red and pink colonies were isolated, tested for adenine requirement and classified as adel or ade2 by complementation tests. I?ilutagenic treatments: (a) EMS. Cells were treated with 3% ethyl methanesulfonate (EMS) i n 0.1 M sodium phosphate buffer (pH 7.2) for one h r at 30" and were then directly plated on selective media. (b) X rays. Cells were irradiated on the surface of plates with 25 krad by means of an OEG-60-T Machlett X-ray tube powered by a custom made X-ray generator (Picker Corp ). (c) UV Cells on the surfaces of the plates were irradiated with 62.4 Jm-2 of UV. Characterimtion of cycl mutat'ons: The nature of the unknown cycl mutants was tested by crossos to two or three well-characterized point mutants, described in Table 1, that span the TABLE 1 T h e cycl point-mutnnt tester alleles Allele
cy c l -I 3 cycI-31 cycl-6
Amino acid position'
____
-1
3 12
cycl-2
21
cycz-72
66
cycl -76 cycl-I23
71 (l02?)
Reference
STEWART et al. 1971 SHERMAN and STEWART 1973,1974 PUTTERMAN, MARGOLIASH and SHERMAN 1974 STEWART et al. unpublished; (see SHERMAN and STEWART 1974) STEWART et al. unpublished; (see SHERMAN and STEWART 1974) STEWART and SHERMAN 1973 SHERMAN et al. 1975
* The numbers refer to the amino acid positions of the corresponding mutational sites that were determined from altered iso-1-cytochromes c in revertants. The cycl-I23 site has been estimated from fine-structure mapping.
787
A MUTABLE GENE I N YEAST
entire gene. The X-ray-induced mitotic recombination frequencies of the heteroallelic diploids were then determined. The transfer of cell suspensions for crossing and testing was facilitated et al. (1974). Cell suspensions of the by the use of inoculating rods as described by SHERMAN heteroallelic diploids were spotted on several lactate plates, and the surfaces of one or two sets of plates were irradiated with 5 krad. The lactate plates were scored after five days of incubation a t 30". Recombination was indicated when increased numbers of colonies appeared over the spotted inocula that were irradiated in comparison to the unirradiated controls. Lack of colonies indicated that recombination did not occur or occurred a t a low rate. A more quantitative measure of recombination rates was occasionally undertaken to verify the spot-test results. Cell suspensions were conveniently prepared free of growth medium and a t a known titre by inoculating the surfaces of 0.5 ml of medium (0.4% Bacto-yeast extract, 0.8% Bacto-peptone, 0.8% glucose and 1% Ionagar) in one-dram vials and, after three days of incubation, suspending the cells in 2 m l of sterile water (SHERMANet a2 1974). Lactate plates were each spread with 0.2 m l of the suspensions from these vials, which contained approximately 1.2 X 107 cells, and two sets d plates were irradiated with 5 krad. The irradiated and unirradiated plates were scored after incubation as indicated above. Characteiization of mete, metl5, ade2, and adel mutants: A similar procedure was used to test for intragenic recombination between unknown met2, m e t l 5 , a h 2 and a d d mutants and appropriate point-mutant tester strains. Although these testers are not as well characterized as the c y c l tester strains, we attempted to choose mutants that would span much of the length of the genes. For m e d 5 , this was done on the basis of previous fine-structure or intragenic complementation maps (SINGHand SHERMAN 1975). RESULTS
The origin and classification of the DELI mutator: During the course of experiments involving the selection of cycl mutants from a variety of strains by the chlorolactate procedure, it was noted that strain D609-2A produced an unusually high frequency of spontaneous cycl mutants. In addition, further investigation revealed that over 95 % of the cycl mutants arising from this and related strains contained multisite mutations encompassing the entire cycl locus; in contrast, such mutations occurred in less than 5 % of the cycl mutants arising from normal strains. Genetic analysis described below demonstrated that the high rate of production of these multisite mutations can be attributed to a single dominant gene designated D E L l . Table 2 summarizes the analysis of a total of 870 spontaneous cycl mutants kolated from 65 different normal (dell+) and mutator ( D E L I ) strains. The cycl mutants were obtained with chlorolactate selection (see MATERIALS AND METHODS) and were classified as either containing or not containing multisite mutations by the various techniques described below. TABLE 2
Isohtion and characterization of cycl mutants f r o m normal and DELI haploid strains Normal dell+
No. haploid strains examined No. c y c l mutants tested No. cycl multisite mutations Percent multisite mutations
28 355 9 3%
Mutator
DELI
37 515 5(17 98%
788
S. L I E B M A N , A. S I N G H A N D F. S H E R M A N
In the early studies, all cycl mutants were actually tested for intragenic recombination with two or three cycl point mutants that map at known positions spanning the entire cycl gene (see MATERIALS AND METHODS). Those mutants that failed to recombine with all of the testers were classified as multisite mutations covering the entire cycl locus. Those mutants that showed recombination were considered to be point mutants or small deletions. Using this classification method, only four multisite mutations were obtained among the 234 cycZ mutants isolated from 14 normal dell+ strains. In contrast, a total of 182 multisite mutants covering the entire cycl locus were found among the 185 cycl mutants isolated from 17 DELI strains. Each of these cycl mutants were also examined for UV sensitivity to see if the mutations extended into the neighboring rad7 locus (see MATERIALS AND METHODS). As expected, none of the “point” mutants were UV sensitive. In contrast most (183 out of 186) of the multisite mutations were sensitive to U V . Furthermore, all of the 182 multisite mutations that arose in DELI strains were UV sensitive, while only one of the four multisite mutations isolated in normal dell + strains was sensitive to UV. In order to further define the size of these multisite mutations, complementation tests with the nearby osml gene were undertaken with a representative number of UV-sensitive multisite mutants (see MATERIALS AND METHODS). All 51 of the UV-sensitive multisite mutants examined, including the one isolated from a normal strain, failed to complement the osml tester. This indicates that all of these mutations extend into at least part of the O S M l locus. In contrast, the three multisite mutants that were not UV sensitive and that were isolated from normal strains complemented osml and therefore did not extend into the OSMl gene. It appears that cycl mutations that arise in DELI strains all extend into at least part of the neighboring RAD7 and OSMl loci. I n contrast, the occasional multisite mutations obtained from normal strains seem to vary in length and do not necessarily include any part of RAD7 or OSMl. These results suggested a simpler method to distinguish the multisite mutations produced in DEL2 strains from the “point” cycl mutants that arise in normal strains. Thus in the subsequent examination of 451 spontaneous cycl mutants isolated from 34 normal and DELI haploid strains, all cycl mutants were tested for UV and osmotic sensitivity. Those cycl mutants that concurrently gained the unselected characteristics of sensitivity to UV and hypertonic medium were considered to contain multisite mutations. The cyci mutants that did not gain these sensitivities were classified as “point” mutants. The classifications of representative cycZ mutants isolated from each parent strain were then further checked by interallelic recombination. I n this way, 325 of 330 cycl mutants isolated from 20 DEL2 strains were found to contain extensive multisite mutations. Of the I21 cycl mutants isolated from 14 normal strains, none contained multisite mutations covering the C Y C I , RAD7 and OSMZ loci. However, five of these mutants were multisite mutations that covered the entire cycl gene, but that did not extend into the RAD7 or OSMZ loci.
A MUTABLE GENE IN YEAST
789
In all of these studies, the distinction between DELI and normal dell+ strains remained clear. In DELI strains, multisite mutations constitute the majority of all spontaneous cycl mutants. These mutations completely encompassed the cycl gene and extended into the neighboring rad7 and osml genes. In dell+ strains, multisitp mutations constitute less than 5% of all spontaneous cycl mutants. The occasional multisite mutations that do arise in dell+ strains do not necessarily cover the RAD7 and OSMl loci. Detailed characteristics of multisite mutations arising in representative DELl strains: To ensure that thc numerous rnultisiie mutations arising in DEL2 strains resulted hom independent events, the chlorolactate procedure was used to isolate cycl mutants from 50 independent subclones of the DELI strain, SL356-4B. One cycl mutant from each subclone was crossed with the three tester strains cycl-23, cycl-2 and cycl-123, which define alterations at positions -1, 21 and 102, respectively, in the 108-residue iso-I-cytochrome c protein. No intragenic recombination was observed in any of these crosses by either the spot or plate tests described in MATERIALS AND METHODS. In addition, each of the 50 mutants failed to complement rad7 and osml strains. Thus, all 50 cycl mutants isolated independently from SL356-4B were multisite mutations that covered the entire cycl gene and exlended into at least part of both the rad7 and osml loci. It was more difficult to determine whether the multisite mutations arising from DEL2 strains also extended into SUP4, anoither gene that is closely linked to cycl (LAWRENCE et al. 1975). The difficulty arises because SUP4 is dominanh so that its inclusion in the mutations could not be established by a simple complementation test. Thus, the approach used was to isolate cycl mutations in DELI stains that contained the UAA suppressor SUP4-o (see MATERIALS AND METHODS for the procedure used to construct the strain). It was reasoned that cycl multisite mutants covering the SUP4 locus would cause the loss of activity of the UAA suppressor. In several multisite mutants, the suppressor activity was lost; in other multisite mutants, the suppressor activity was retaind. These results prove that the multisite mutations do not always cover SUP4. However, since the SUP4-o marker was found to be unstable on chlorolactate medium, it is not clear whether any of the multisite mutations really cover the SUP4 locus. Anather approach to determining whether SUP4 is encompassed by the multisite mutations arising in DELI strains was to' select for loss of the UAA-suppressor activity in a DELI SUP4-o strain and to see if any other neighboring genes were also lost, thus indicating the presence of multisite mutations. This test was accomplished with the help of a UAA marker, cad-100, also present in the DEL? SUP4-o strain. The cad-100 marker ordinarily causes resistance to canavanine; holwever, in the presence of SUP4-0, the canl-100 marker is suppressed and the strain is sensitive to canavanine. Thus, by selecting for revertants that are resistant to canavanine, we were able to select for lass of the SUP4-o Suppressor. Out of 30 revertants that had lost the SUP4-o p h e n w e , none were UV or osmotic sensitive. Thus, there is no evidence that the SUP4 revertants induced in the DELI strain contained multisite mutations. To further characterize the nature of the multisite mutations, we attempted
790
S. LIEBMAN, A. SINGH A N D F. SHERMAN
to revert a representative multisite mutation. In order to eliminate the occurrence of recessive mutations that cause growth on lactate medium by increasing the et al. 1974), tests of revertibility were level of iso-2-cytochrome c (SHERMAN performed on a diploid strain homozygous for one of the multisite mutations generated in a DELI strain. The diploid strain was treated with UV, X rays and and approximately 4.4 X 10' EMS as described in MATERIALS AND METHODS, treated and untreated cells were plated on lactate medium. No revertants were observed, which is consistent with the conclusion that the multisite cycl mutants arising from DELl strains are extensive deletions or are other gross chromosomal aberrations. Action of DELl on other genes: Experiments were undertaken in order to determine whether DELI is a general mutator that can act on various regions of the yeast genome. Taking advantage of the red color of adel and ade2 mutants and of the methyl mercury resistance of met2 and met15 mutants, and using the nystatin enrichment technique, spontaneous mutants were isolated in the DELI strain, SL3564B, and examined for the nonrevestible, multisite mutations. As described above, 50 independently derived cycl mutants isolated frcm different subclones of SL3564B were all found to contain multisite mutations of the cycl region. In contrast, none of the five met2, 31 metI5, 26 a b 2 and 31 adel independent mutations isolated from this DELI strain were classified as multisite mutants. Each of these mutants, except for two that were too leaky to1 be tested, showed either intragenic recombination with tester strains or could be reverted to the apparent wild-type state. Thus, there is no evidence that DELl acts on regions other than the region encompassing the CYCI locus. However, the possibility that DELI can act on regions other than those tested or that multisite mutations produced at the ade acd met loci were not detected because they are lethal can not be eliminated. Rates of adel arid ade2 mutations: As part of the comparative study of spontaneous mutation rates in DELI strains, the rates of adel and ade2 mutations were calculated from data of the last 38 nystatin enrichment experiments per€ormed on the DELI strain, SL3564B (see MATERIALS AND METHODS for experimental details). Mutation rates, a, were obtained from the equations Po," ln(m) = 1.24 and a = m (ln2)/N (LEAand COULSON 1949) and from r = m ln(Cm) and a = m (ln2)/N (LURIAand DELBRUCK 1943), where: a = mutation rate (mutations per cell per generation) ;N = final number of cells per culture; m = the number of mutations per culture; C = number of cultures; I, = the number of mutants in the median culture; and r = t h c average number of mutants per culture among C cultures. In the calculations, we used our experimental values of C=38, N=7.5 x l o 7 and the observed median ( r , ) and average ( r ) number of adel and ade2 mutants per culture. The experimental values of r and r, and the calculated values of a are shown in Table 3. The results indicate that the spontaneous forward mutation rates to adel and ade2 in the DELI strain, SL356-4B, are approximately 5 x 10-9 to 8 X 1P9. Rates of cycl mutation in DELl and normal strains: The forward mutation rates to cycl in the DELI strains SL356JI.B and SL392-2B were compared with
791
A MUTABLE GENE IN YEAST
TABLE 3 Forward mutaticil rates to adel and ade2 in the DELI strain, SL356-4B r
adel ade2
1.82 1.55
a*
5.5 x 10-9 x 10-9
4.8
nt
To
1 1
8.3 8.3
x 10-9 x 10-9
* a was calculated from the formulas r = m ln(Cm) (LURIAand DELBRUCK 1943) and a = m (ln2)/n, where N = 7.5 x IO7 and C = 38. + a was calculated from the formulas r , / m - ln(m) = 1.24 (LEAand COULSON1949) and a = m (ln2)/N, where N = 7.5 x 107.
the cycl mutation rates in the normal strains D311-3A and D911-2B. A variety of methods were used to estimate these rates. The mutation rate to cycl in D911-2B was obtained using the Luria-Delbruck (LURIAand DELBRUCK 1943) fluctuation test. D911-2B was subcloned on complete media, and 55 colonies, each containing l o s cells, were individually suspended in water and spread o n 9-cm diameter agar plates containing chlorolactate medium. Henceforth, the number of cells per plate was determined by quantitatively washing cells off the plates. Within two days, the number of cells per plate increased to 1.8 x 1Os, where it remained constant for an additional several days until the resistant colonies began to appear. Chlorolactate-resistant colonies were then picked and tested to determine if they were cycl mutants. As soon as one cycl mutation was found from a given plate, additional chlorolactate-resistant mutants from that plate were no longer tested. Plates that did not give any cycl mutations were picked and tested exhaustively. Of the 65 cycl mutations obtained from D911-2B, less than 5% were multisite mutations. Among the 55 plates examined, 16 did not contain any cycl mutants, while 39 plates contained one or more cycl mutants. Using the equations po = cmand a = m (ln2)/Nt-N0 LURIAand DELBRUCK (1943), where p. is the fraction of plates that do not have any cycl mutants and is equal t o 0.29 in the present experiment; m is the average number of cycl mutations that occurred per culture; N t is the final cell count per plate and is equal to 1.8 f 0.1 X lo8 in the present experiment; N o is the initial number of cells per colony on complete media and is equal to one in the present experiment; and a is the mutation rate, mutations per cell per generation, we calculate the mutation rate to be 4.8 x l k9. Attempts to use the same fluctuation methold to measure the mutation rate in DELI strains failed because all of the plates contained cycl mutants, apparently due to an increased mutation rate. We were unable to reduce the number of colonies because the background growth led to a minimum of 1.1 x lo8 cells per 9-cm diameter plate, no matter how many cells were initially placed on the plate. Even when 1.4-cm diameter wells were used instead of plates, background growth led to a titer 04 approximately 2 x lo6 cells per well, and all of 54 wells contained one or more cycl mutant colonies. Because of these problems, the mutation rate in the DELI strain SL392-2B was measured by an adaptation of the LURIAand DELBRUCK (1943) method. To determine the mutation rate, l o 4
792
S. LIEBMAN, A. SINGH A N D F. SHERMAN
cells from an overnight culture grown in synthetic lactate medium were spread on each of 37 agar wells containing chlorolactate medium. Due to the low frequency of cycl mutants in the culture, it is likely that none of the cells plated were cycl mutants. Within three days, the number of cells per well increased to 1.6 0.4 x IO6, where it remained constant for an additional several days until resistant colonies began to appear. Thus, each cell gave rise to a microcolony containing on the average 160 cells. After further incubation, an average of 6.8 rf: 2.5 cycl mutant colonies arose on each well. Over 95% of these mutants were of the multisite type. Since virtually all of the mutants arose during the formation of the microcolonies, po = 1-6.8 x IO-* and m is approximately equal to 6.8 x Therefore a, the mutation rate per cell per division, was deduced to be approximately 2.9 1.7 x In another approach, mutation rates were estimated by using the principle that when a population equilibrium is reached between mutant and normal cells during exponential gromh in a medium totally selective against the mutant, the mutant frequency is then approximately equal to the mutation rate (OGURet al. 1959). These conditions were met by growing strains in liquid lactate medium where normal CYCl+, but not cycl mutant, cells can grm. Thus, the mutation rates of cycl were simply determined from the frequencies of cycl mutants in an equilibrium population of cells grown in lactate medium. The frequencies o€ cycl mutants were estimated using the chlorolactate selection procedure. The results of several experiments with normal and DEL2 strains are presented in Table 4. The normal dell+ strain, D311-3A, was kept in an exponential phase of growth in lactate medium by periodically transferring cells to fresh medium. After four days of exponential growth, 4.4 x IO6 cells per plate were spread on chlorolactate medium. Within three days, the number of cells per plate increased to 1.1 e 0.1 x lo8. Then the number of cells remained constant for an additional several days until the resistant colonies began to appear. Among the 19 plates examined, a total of 546 resistant colonies appeared. Complementation tests indicated that only 2% of these colonies were cycl mutants. This frequency A indicates that the cycl mutation rate in D311-3A is approximately 5 X
*
*
TABLE 4 Rates of cycl mutations in normal (dell f)and DELl strains Strain X o .
Genotype
Days of growth*
D311-3A D311-3A SL356-4B SL3564.B SL356-4B SL3564B
dell+ dell + DELl DELl DELl DELl
4 7 2 5 5 5
No. of cells per plate:
No. of
3 days
plates
Initial
4.3~106 1.1~108 4.4~106 1 . 1 ~ 1 0 8 1x105 5 . 5 ~ 1 0 5 1.9X107 4.2X107 5.7X106 5.7X1O7 4.7X105 4.7x1O6
19 20 5 3 2
2
Mutants per plate
29 29 6 358 225 40
% cycf mutants
Rate of cycf mutations
2 2 100 100
5x1e9 5x10-Q 11X10-6 9XIW 4X1W 9x10-6
100 100
* Day of exponential growth in liquid lactate medium before plating on chlorolactate plates. The number of cells initially plated increased to approximately 108 per plate due to residual growth. When plating SL3564B, this number was constrained by the addition of other cells.
793
A M U T A B L E G E N E IN YEAST
duplicate determination was made in a similar manner after the exponential culture of D311-3A was grown for an additional three days on lactate medium. None of these mutants were Again the mutation rate was estimated at 5 x multisite mutations. The cycl mutation rate in the DELl strain, SL3564B was estimated by a similar procedure. Here, however, when the background growth of SL3564B on the chlorolactate plates was permitted to reach the limiting level of 1.1 X 10' cells per plate, the resistant colonies were too numerous to score. In order to reduce the background level of SL3564B, various amounts of the normal diploid strain D-261 were added to the chlorolactate plates. These added cells reduce the background growth of SL3564B, since a chlorolactate plate can support growth only to a total of approximately 1-2 x 108 cells. The level of background growth of plates spread with various amounts of D-261 was determined by qwmtitatively washing cells off of the chlorolactate plates and counting them under the microscope. The diploid cells gave rise to virtually no resistant colonies, presumably because most chlorolactate resistant mutations are recessive. In order to measure the cycl mutation frequency in SL356-4B, plates were spread with both D-261 and SL3564B at a ratio of at least, respectively, ten to one. Under these conditions, it is assumed that D-261 will grow to approximately the same level that it reached in the absence of SL356-4.B. It is also assumed that SL356-4.B will increase by the same factor as D-261. The results of four determinations of the cycl mutation rate in the DELl strain SL356-4B are shown in Table 4.The cycl mutation rate is between IO4 and and nearly all of these mutants are multisite mutations. These results, summarized in Table 5, show that the forward mutation rate to cycl is increased by approximately three orders of magnitude in the DELl strains SL392-2B and SL3564B as compared with the normal strains D311-3A and D911-2B. It should be noted that the mutation rate determinations were quite consistent, even though a variety of different strains and methods were used. Since nearly all of the cycl mutants produced in the DELl strains are multisite mutations, while none or very few cycl multisite mutations are produced in normal strains, it is clear that the DELl gene increases the frequency of mutation to cycl multisite mutations by four or five orders of magnitude. In contrast, the forward mutation rates to adel and ade2 estimated in a DELI strain are of TABLE 5 Summary of mutation rate determinations to cycl in normal (dell+) and DELl strains Strain No.
D311-3A D911-2B SL356-4B SL392-2B
Genotype
dell + dell + DELl DELl
Rate of mutations to cycl
5 x 10-9" 4.8 x 10-9t 8 (+. 3) x le6* 2.9 (f1.7) X le6+
Rate of cycl multisite mutations
